ebi is an outstation of the european molecular biology laboratory. uniprotkb sandra orchard
TRANSCRIPT
EBI is an Outstation of the European Molecular Biology Laboratory.
UniProtKB
Sandra Orchard
Importance of reference protein sequence databases
• Completeness and minimal redundancy
A non redundant protein sequence database, with maximal coverage including splice isoforms, disease variant and PTMs.
Low degree of redundancy for facilitating peptide assignments
• Stability and consistency Stable identifiers and consistent nomenclature
Databases are in constant change due to a substantial amount of work to improve their completeness and the quality of sequence annotation
• High quality protein annotation
Detailed information on protein function, biological processes, molecular interactions and pathways cross-referenced to external source
Summary of protein sequence databases
Database Description Species
UniProtKB Expertly curated section (UniProtKB/Swiss-Prot) and computer-annotated section (UniProtKB/TrEMBL); minimum level of redundancy; high level of integration with other databases; stable identifiers; diversity of sources including large scale genomics, small scale cloning and sequencing, protein sequencing, PDB, predicted sequences from Ensembl and RefSeq
Many
UniRef100 Assembled from UniProtKB, Ensembl and RefSeq; merges 100% identical sequences; stable identifiers
Many
Ensembl Predictions using automated genome annotation pipeline; explicitly linked to nucleotide and protein sequences; stable reference; merge their annotations with Vega annotations at transcript level; extensive quality checks to remove erroneous gene models ; high level of integration with other databases
Over 50 Eukaryotic genomesEnsembl Genomes: Metazoa, Plants and Fungi, Protists, Bacteria and Archaea
RefSeq NCBI creates from existing data; ongoing curation; non-redundant; explicitly linked nucleotide and protein sequences; stable reference; high level of integration with other databases
Limited to fully sequenced organisms
Entrez protein (NCBInr) Assembled from GenBank and RefSeq coding sequence translations and UniProt KB ; annotations extracted from source curated databases; high degree of sequence redundancy
Many
Updated from Nesvizhskii, A. I., and Aebersold, R. (2005) Interpretation of shotgun proteomic data: the protein inference problem. Mol. Cell. Proteomics. 4,1419–1440l
UniProtKB
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UniProt Knowledgebase: 2 sections
1. UniProtKB/Swiss-Prot Non-redundant, high-quality manual annotation - reviewed
2. UniProtKB/TrEMBL Redundant, automatically annotated - unreviewed
www.uniprot.org
Sequence Sequence features
Ontologies
ReferencesNomenclature
Splice variants
Annotations
UniProtKB
Manual annotation of UniProtKB/Swiss-Prot
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Sequence curation, stable identifiers, versioning and archiving
For example – erroneous gene model predictions, frameshifts….
..premature stop codons, read-throughs, erroneous initiator methionines…..
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Splice variants
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Identification of amino acid variants
..and of PTMs
… and also
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Domain annotation
Binding sites
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Protein nomenclature
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Controlled vocabularies used whenever possible…
Annotation - >30 defined fields
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..and also imported from external resources
Binary interactions taken from the IntAct database
Interactors of human p53
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Controlled vocabulary usage increasing – for example from the Gene Ontology
Annotation for human Rhodopsin
1 Evidence at protein levelThere is experimental evidence of the existence of a protein
(e.g. Edman sequencing, MS, X-ray/NMR structure, good quality protein-protein interaction , detection by antibodies)
2 Evidence at transcript levelThe existence of a protein has not been proven but there is expression data (e.g. existence of cDNAs, RT-PCR or Northern blots)
that indicates the existence of a transcript.
3 Inferred from homologyThe existence of a protein is likely because orthologs exist in closely related species
4 Predicted
5 Uncertain
Sequence evidence
Type of evidence that supports the existence of a protein
Manual annotation of the human proteome(UniProtKB/Swiss-Prot)
• A draft of the complete human proteome has been available in UniProtKB/Swiss-Prot since 2008
• Manually annotated representation of 20,231 protein coding genes with 36,865 protein sequences - an additional 33,243 UniProtKB/TrEMBL form the complete proteome set
• Approximately 67,600 single amino acid polymorphisms (SAPs), mostly disease-linked
• ~75,500 post-translational modifications (PTMs)• Close collaboration with NCBI, Ensembl, Sanger Institute
and UCSC to provide the authoritative set to the user community
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Searching UniProt – Simple Search
• Text-based searching• Logical operators ‘&’ (and), ‘|’
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Searching UniProt – Advanced Search
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Searching UniProt – Search Results
Each linked to the UniProt entry
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Searching UniProt – Search Results
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Searching UniProt – Search Results
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Searching UniProt – Blast Search
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Searching UniProt – Blast Search
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Searching UniProt – Blast Results
Alignment with query sequence
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Searching UniProt – Blast Results
UniProtKB/TrEMBL
Multiple entries for the same protein (redundancy) can arise in UniProtKB/TrEMBL due to:
o Erroneous gene model predictionso Sequence errors (Frame shifts)o Polymorphismso Alternative start siteso Isoforms
Apart from 100% identical sequences all merged sequences are analysed by a curator so they can be annotated accordingly.
Why do we need predictive annotation tools?
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UniProtKB
UniProtKB/Swiss-Prot
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• Automated clean-up of annotation from original nucleotide sequence entry
• Additional value added by using automatic annotation
• Recognises common annotation belonging to a
closely related family within UniProtKB/Swiss-Prot
• Identifies all members of this family using pattern/motif/HMMs in InterPro
• Transfers common annotation to related family members in TrEMBL
Automatic Annotation
← Name (non-standard)
← Taxonomy
← Publication
← Sequence
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InterPro
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Finding a complete proteome in UniProtKB
Complete Proteomes
MS Proteomics
• Require each sequence (inc isoforms) to be present in the dataset as an separate entity for search engines to access
• For higher organisms, with isoforms, expanded set made available on ftp site
• Fasta files by FTP• One file per species containing canonical + isoform sequences
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